The role of the receptor tyrosine kinase RET as a dependence receptor during enteric nervous system formation
Enteric neurons are derived from vagal and sacral neural crest cell populations. Using human embryonic tissue aged 4-14 weeks of gestation the timing and pattern of gastrointestinal colonisation by vagal neural crest cells, and the development of interstitial cells of Cajal and the smooth musculature of the gut, were investigated using immunohistochemistry. Formation of both the enteric nervous system and smooth muscle involves rostrocaudal migration and maturation of these tissues. The gut is fully colonised by neural crest cells at week 7 and these cells first form the myenteric plexus external to the future circular muscle layer. Secondary centripetal migration to the submucosal plexus occurs at week 8 in the oesophagus and week 11 in the midgut. Smooth muscle appears at week 8, first as the circular muscle layer, with secondary formation of the longitudinal layer. ICCs are present from week 8 and develop in close association with the myenteric plexus in the midgut and hindgut. At week 14 all components required for a mature gut are present. Rearranged during transfection (RET), a tyrosine kinase receptor and the most commonly linked gene to the enteric nervous system developmental disorder, Hirschsprung's disease, has been described in vitro as a dependence receptor, i.e.: expression of the receptor causes a cellular dependency on its ligand. In order to test whether RET functions as a dependence receptor in vivo constructs designed to perturb the caspase dependent cleavage of RET were introduced by electroporation of neural crest in E1.5 chicken embryos. Inhibition of Caspase-9 dependent apoptosis reveals a role for cell death in early control of neural crest cell numbers. No effect is observed when intracellular wildtype RET is introduced. However, introduction of a mutated form of RET induces a delay in the migration and differentiation of enteric precursors. These observations, combined with Tunel and ret in situ data, suggest a role for RET as a dependence receptor in vivo via a caspase-9 dependent mechanism.